Role of the mitochondrial Bol1 and Bol3 proteins in iron-sulfur cluster delivery to diverse recipient proteins

线粒体铁硫（Fe/S）蛋白的生物发生由保守的ISC组装机制催化，可分为三个主要步骤。首先，硫供体Nfs1-Isd11-Acp1和其他四个ISC因子在支架蛋白Isu1上重新合成[2Fe-2S]簇。其次，Fe/S簇通过专用的Hsp70伴侣系统从Isu1中释放出来，并通过单硫醇glutaredoxin Grx5转移到[2Fe-2S]靶蛋白上。第三，瞬时grx5结合的[2Fe-2S]簇被Isa1-Isa2-Iba57蛋白转化为[4Fe-4S]簇，该簇被特异性ISC靶向因子Nfu1和Ind1瞬时结合，最终插入受体[4Fe-4S]蛋白。该项目的工作已经确定了两种酵母线粒体蛋白Bol1和Bol3作为额外的ISC靶向因子，协同促进脂酰合成酶和呼吸复合物II的特异性成熟。人类BOLA3 （Bol3同源物）而不是BOLA1具有类似的靶向功能。与L. Banci合作，对这两种BOLA蛋白的溶液结构进行了解析。生化研究表明，这两种BOLA复合物与[2Fe-2S]簇结合GLRX5（人类Grx5同源物）的亲和性不同，但这一观察结果的生理意义尚不清楚。突变分析表明，[2Fe-2S]簇与BOLA1- glrx5配合物结合，独立于BOLA1中三个保守的His残基，使得holo-配合物的确切结构开放。我们的工作提出了一个核心问题，即为什么[4Fe-4S]蛋白质组装需要含有[2Fe-2S]簇的Bol-Grx5复合体。此外，基于酵母和人类细胞（以及疾病）中完全不同的BOL和GRX5缺失表型，很明显，在复杂的Fe/S蛋白组装途径中，BOL - GRX5的确切作用位点仍然是开放的。在下一个资助期，我们的目标是对Bol蛋白功能进行生理学上有意义的机制理解，以解释[4Fe-4S]簇插入线粒体载脂蛋白的参与。我们将通过检查Bol蛋白的功能和相互作用的伙伴蛋白来测试Bol蛋白在氧化应激条件下是否表现出增加的重要性。我们将在体外重建脂酰合成酶成熟，以精确定义Bol和Grx5蛋白的机制功能。为了支持这一目标，boll - grx5配合物的晶体结构将得到解决。最后，我们将进行细胞生物学和生化研究，以阐明Bol-Grx5复合物是在Isa-Iba57蛋白之前还是之后起作用。全面了解Bol蛋白功能的机制对于bola3相关线粒体疾病MMDS2的分子观点非常重要。

The biogenesis of mitochondrial iron-sulfur (Fe/S) proteins is catalyzed by the conserved ISC assembly machinery and can be dissected into three major steps. First, a [2Fe-2S] cluster is synthesized de novo on the scaffold protein Isu1 by the sulfur donor Nfs1-Isd11-Acp1 and four other ISC factors. Second, the Fe/S cluster is released from Isu1 by a dedicated Hsp70 chaperone system, and transferred via the monothiol glutaredoxin Grx5 to [2Fe-2S] target proteins. Third, a transiently Grx5-bound [2Fe-2S] cluster is converted by the Isa1-Isa2-Iba57 proteins to a [4Fe-4S] cluster, which is transiently bound by the specific ISC targeting factors Nfu1 and Ind1, and finally inserted into recipient [4Fe-4S] proteins. Work within this project has identified the two yeast mitochondrial proteins Bol1 and Bol3 as additional ISC targeting factors that cooperatively facilitate specific maturation of lipoyl synthase and respiratory complex II. Human BOLA3 (Bol3 homolog) but not BOLA1 performs a similar targeting function. The solution structure of the two BOLA proteins was resolved in cooperation with L. Banci. Biochemical studies revealed rather different affinities of the two BOLA complexes with [2Fe-2S] cluster-bound GLRX5 (human Grx5 homolog), yet the physiological meaning of this observation remains unclear. Mutational analysis indicated that the [2Fe-2S] cluster is bound to the BOLA1-GLRX5 complex independently of three conserved His residues in BOLA1, leaving the exact architecture of the holo-complex open. Our work has raised the central question of why a [2Fe-2S] cluster-containing Bol-Grx5 complex is needed for [4Fe-4S] protein assembly. Further, based on the radically different BOL and GRX5 deletion phenotypes in both yeast and human cells (and in diseases), it became evident that the precise site of action of Bol-Grx5 within the complex Fe/S protein assembly pathway remains open. In the next funding period, we aim to reach a physiologically meaningful mechanistic understanding of Bol protein function that explains the involvement in [4Fe-4S] cluster insertion into mitochondrial apoproteins. We will test whether the Bol proteins exhibit an increased importance under oxidative stress conditions by examining their function and interacting partner proteins. We will reconstitute lipoyl synthase maturation in vitro to precisely define the mechanistic function of the Bol and Grx5 proteins. In support of this aim, the crystal structure of the Bol-Grx5 complexes will be solved. Finally, we will perform cell biological and biochemical studies to elucidate whether the Bol-Grx5 complexes act before or after the Isa-Iba57 proteins. A comprehensive mechanistic understanding of Bol protein function will be important for a molecular view of the BOLA3-associated mitochondrial disease MMDS2.